Monoclonal antibodies to PACAP

ABSTRACT

Disclosed are a monoclonal antibody having affinity for PACAP, a partial peptide thereof, a precursor thereof or VIP; a hybridoma cell which produces the above monoclonal antibody; and an immunoassay for assaying PACAP by a competitive method or a sandwich method using the above antibody, whereby PACAP can be specifically detected with high sensitivity.

BACKGROUND OF THE INVENTION

The present invention relates to an antibody which is novel and usefulin that it has specific affinity for PACAP, and more particularly to anantibody useful for development of assays of PACAP on the basis ofantigen-antibody reactions or for diagnosis and treatment of diseasesrelated to PACAP.

Various hormones secreted by brain hypothalami and hypophyses have beenknown. Examples thereof include thyrotropin releasing hormone,luteinizing hormone releasing hormone, somatostatin, adrenocorticotropichormone, growth hormone and prolactin. Action thereof has been studiedin detail. Recently, a novel bioactive substance of hypothalamic originother than these hormones was studied based upon adenylate cyclaseactivity, and consequently a peptide consisting of 38 amino acidresidues which had not been reported till then was discovered from sheephypothalami. This peptide was named "PACAP38NH₂ " and has a structurerepresented by the following formula (SEQ ID NO:2):

    His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn Lys-NH.sub.2

It was disclosed in applications for patents (Japanese PatentApplication Nos. 1-155791/1990 and 1-284771/1990) on cDNA of sheepPACAP38, and an application for a patent (Japanese Patent ApplicationNo. 1-259924/1990) on the partial structure of cDNA of human PACAP38that the amino acid sequence of the mature portion of sheep PACAP38 wasthe same as that of human PACAP38, and that some amino acids of theprecursors thereof were substituted. It is deduced from the position ofcontinuous basic amino acids shown in the cDNA sequence of PACAP38NH₂that PACAP27NH₂, in addition to PACAP38NH₂, will exist as a peptide cutout of the precursor.

In fact, according to subsequent studies PACAP27NH₂ was also isolatedfrom sheep hypothalami, in addition to PACAP38NH₂. The structure thereofis represented by the following formula (SEQ ID NO:2):

PACAP27NH₂

    His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu-NH.sub.2

PACAP38NH₂ and PACAP27NH₂ are hereinafter represented by the generalterm of "PACAP". The 28 amino acid residues on the N-terminal side ofPACAP38NH₂ containing PACAP27NH₂ show 68% homology with vasoactiveintestinal polypeptide (VIP) well known as a brain-gut peptide. However,it has been reported that the adenylate cyclase activating activity ofPACAP exceeds at least 1,000 times that of VIP.

Thus, the action of PACAP is anticipated to be different from that ofVIP, and a deep interest is taken in the physiological role thereof andthe relation thereof to the pathology.

Although the interest in PACAP is increased as described above, basicphysiological information such as existing sites other than hypothalamiof PACAP and a plasma level thereof is scarcely obtained, and therelation thereof to the pathology is also unknown. This is mainly causedby that any monoclonal antibodies specifically recognizing PACAP havehitherto not been prepared and that any immunoassays for assaying PACAPspecifically and highly sensitively have not been developed. Theseimmunological procedures are considered to be one of the most effectivemeans to study PACAP, particularly the metabolic pathways thereof, thesecretory mechanism thereof, the receptor system thereof, the relationthereof to the pathology and the like collectively. The establishment ofthese procedures has therefore been earnestly desired in various fields.

Previously, competitive radioimmunoassays (RIA) generally using one kindof antibody and enzyme immunoassays (EIA) have been developed andemployed to assay low molecular weight peptides such as PACAP. On theother hand, sandwich immunoassays using two kinds of antibodies have theadvantages of (1) improving the specificity of assay systems because ofthe use of two kinds of antibodies and (2) being little affected bynonspecific interfering factors because of the use of the antibodies inlarge excess to substances to be assayed. Until now, however, it hasbeen unknown at all whether or not the low molecular weight peptideshaving no disulfide linkage such as PACAP can be assayed with highsensitivity by the sandwich methods. Namely, in case of the lowmolecular weight peptides having no disulfide linkage such as PACAP, thepossibility is conceivable that binding sites of two kinds of antibodiesare in so close proximity to each other as to exert influences such assteric hindrance, which results in difficulty of the establishment ofhighly sensitive sandwich methods.

SUMMARY OF THE INVENTION

The present inventors prepared polyclonal and monoclonal antibodieshaving affinity for PACAP and having different reaction specificity forpartial peptides of PACAP and VIP, and developed an immunoassay whichcan specifically detect PACAP with high sensitivity using the antibodiesand which can fractionate and determine PACAP38NH₂ and PACAP27NH₂.

In accordance with the present invention, there are provided amonoclonal antibody having affinity for PACAP, a partial peptide ofPACAP, a precursor of PACAP or VIP; a hybridoma cell which produces theabove monoclonal antibody; and an immunoassay for assaying PACAP by acompetitive method or a sandwich method using the above antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing antibody titer to PACAP38NH₂ in mouseantisera;

FIG. 2 is a graph showing antibody titer to PACAP(11-27)NH₂ in rabbitantisera;

FIGS. 3(a)-(f) are a series of graphs showing the results assayed forPACAP38 and related peptides by a competitive method-enzyme immunoassayusing antibodies of the present invention, thereby clarifyingrecognition sites of the antibodies of the present invention, FIG. 3(a)shows the results using antibody PA-1Na. FIG. 3(b) shows the resultsusing antibody PA-3Na. FIG. 3(c) shows the results using antibodyPA-5Na. FIG. 3(d) shows the results using antibody PA-6Na. FIG. 3(e)shows the results using antibody PA-2Ca. FIG. 3(f) shows the resultsusing antibody PA-1Ca;

FIGS. 4 to 6 are graphs showing the results detected for PACAP38NH₂ bysandwich method-enzyme immunoassays using antibodies of the presentinvention, FIG. 4 shows the results using PA-6Na F(ab')₂ -HRP. FIG. 5shows the results using PA-1Ca F(ab')₂ -HRP. FIG. 6 shows the resultsusing PA-2Ca IgG-HRP.

FIG. 7 is a graph showing the reactivity of PACAP and related peptidesin the sandwich method-enzyme immunoassay of the present invention;

FIGS. 8 to 11 are graphs showing the reactivity of PACAP27NH₂ andrelated peptides in the sandwich method-enzyme immunoassays, FIG. 8shows the results using PA-1Na. FIG. 9 shows the results using PA-3Na.FIG. 10 shows the results using PA-5Na. FIG. 11 shows the results usingPA-6Na; and

FIG. 12 is a graph showing the results of examination of theneutralization activity of anti-PACAP antibodies to PACAP38NH₂ by theuse of cultured cells.

FIG. 13 shows immunoblot analysis of the products of E. coli transfectedwith the expression plasmid containing human prepro-PACAP cDNA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As the partial peptides of PACAP, any peptides may be used as long asthey have partial sequences of PACAP. Examples of such peptides includepeptides corresponding to an N-terminal portion of PACAP, such asPACAP(1-13) having the following sequence (SEQ ID NO:3):

    His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr,

peptides corresponding to a region from the N-terminal portion to acentral portion of PACAP, such as PACAP(4-27) having the followingsequence (SEQ ID NO:4):

    ______________________________________                                        Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln Met                       Ala Val Lys Lys Tyr Leu Ala Ala Val Leu,                                      ______________________________________                                    

peptides corresponding to a region from a C-terminal portion to acentral portion of PACAP38NH₂, such as PACAP(14-38) having the followingsequence (SEQ ID NO:5):

    ______________________________________                                        Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu                       Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn Lys,                                  ______________________________________                                    

peptides corresponding to the C-terminal portion of PACAP38NH₂, such asPACAP(31-38) having the following sequence (SEQ ID NO:6):

    Tyr Lys Gln Arg Val Lys Asn Lys, and

peptides corresponding to a region from a central portion to aC-terminal portion of PACAP27, such as PACAP(11-27) having the followingsequence (SEQ ID NO:7):

    ______________________________________                                        Ser Arg Tyr Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala                       Ala Val Leu--NH.sub.2                                                         ______________________________________                                    

Of these peptides, it is preferred that the C-termini of PACAP(31-38)and PACAP(11-27) have amide forms, and the C-termini of the otherpeptides may have either amide forms or free carboxylic acid forms. Theamide form is represented by giving NH₂ as PACAP(31-38)NH₂, and the freecarboxylic acid form is given no symbol or represented by giving OH, forexample, PACAP(31-38)OH.

Examples of a precursor of PACAP include a precursor of human PACAPconsisting of an amino acid sequence represented by the followingformula (SEQ ID NO:8) or a portion thereof:

    ______________________________________                                             Met    Thr    Met   Cys  Ser  Gly  Ala  Arg  Leu                         Ala  Leu    Leu    Val   Tyr  Gly  Ile  Ile  Met  His                         Ser  Ser    Val    Tyr   Ser  Ser  Pro  Ala  Ala  Ala                         Gly  Leu    Arg    Phe   Pro  Gly  Ile  Arg  Pro  Glu                         Glu  Glu    Ala    Tyr   Gly  Glu  Asp  Gly  Asn  Pro                         Leu  Pro    Asp    Phe   Gly  Gly  Ser  Glu  Pro  Pro                         Gly  Ala    Gly    Ser   Pro  Ala  Ser  Ala  Pro  Arg                         Ala  Ala    Ala    Ala   Trp  Tyr  Arg  Pro  Ala  Gly                         Arg  Arg    Asp    Val   Ala  His  Gly  Ile  Leu  Asn                         Glu  Ala    Tyr    Arg   Lys  Val  Leu  Asp  Gln  Leu                         Ser  Ala    Gly    Lys   His  Leu  Gln  Ser  Leu  Val                         Ala  Arg    Gly    Val   Gly  Gly  Ser  Leu  Gly  Gly                         Gly  Ala    Gly    Asp   Asp  Ala  Glu  Pro  Leu  Ser                         Lys  Arg    His    Ser   Asp  Gly  Ile  Phe  Thr  Asp                         Ser  Tyr    Ser    Arg   Tyr  Arg  Lys  Gln  Met  Ala                         Val  Lys    Lys    Tyr   Leu  Ala  Ala  Val  Leu  Gly                         Lys  Arg    Tyr    Lys   Gln  Arg  Val  Lys  Asn  Lys                         Gly  Arg    Arg    Ile   Ala  Tyr  Leu                                        ______________________________________                                    

The present inventors made various investigations to produce themonoclonal antibodies and the polyclonal antibodies to PACAP. As aresult, the antibodies roughly classified into 5 classes were obtained.

The antibodies classified as class I recognize the N-terminal portion ofPACAP. Namely, they react with PACAP38NH₂, PACAP27NH₂, PACAP(1-13) andPACAP(4-27), and do not react with PACAP(14-38)NH₂ and PACAP(31-38)NH₂.

The antibodies classified as class II recognize the region from theN-terminal portion to the central portion of PACAP. Namely, they reactwith PACAP38, PACAP27NH₂ and PACAP(4-27), and do not react withPACAP(1-13), PACAP(14-28)NH₂ and PACAP(31-38)NH₂.

The antibodies classified as class III recognize the region from theC-terminal portion to the central portion of PACAP. Namely, they reactwith PACAP38NH₂ and PACAP(14-38)NH₂, and do not react with PACAP27NH₂,PACAP(1-13), PACAP(4-27) and PACAP(31-38)NH₂.

The antibodies classified as class IV recognize the C-terminal portionof PACAP38NH₂. Namely, they react with PACAP38NH₂, PACAP(14-38)NH₂ andPACAP(31-38)NH₂, and do not react with PACAP27NH₂, PACAP(4-27) andPACAP(1-13).

The antibodies classified as class V recognize the C-terminal portion ofPACAP27NH₂. Namely, they are antibodies to PACAP(11-27)NH₂.

The antibodies classified as class I are further classified into classIa and class Ib. The antibodies classified as class Ia exhibit only across reactivity with VIP of less than 0.5%, and the antibodiesclassified as class Ib exhibit a cross reactivity with VIP of 0.5% ormore.

Many of the antibodies classified as class II show only a crossreactivity with VIP of less than 0.5%, and many of the antibodiesclassified as classes III to V exhibit only a cross reactivity with VIPof less than 0.01%.

These antibodies can be used in ordinary tissue staining or competitiveimmunoassays. The present inventors further made various investigationsto develop an excellent immunoassay, and consequently developed thesandwich immunoassay in which to kinds of these monoclonal or polyclonalantibodies are used in combination.

In the immunoassay for assaying PACAP by the sandwich method of thepresent invention, each of the antibodies used in a primary reaction (areaction of an antibody for a solid phase with a substance to be tested)and a secondary reaction (a reaction of a labeled antibody with thesubstance to be tested) may be either the polyclonal antibody or themonoclonal antibody. It is however preferred that one of them is theantibody which recognizes the N-terminal portion of PACAP38NH₂ (classI), the region from the N-terminal portion to the central portion (classII), the region from the C-terminal portion to the central portion(class III) or C-terminal portion (class IV) and the other is theantibody which recognizes regions other than the regions describedabove.

Namely, the present inventors discovered that PACAP38NH₂ was detectedwith high sensitivity in combinations of class I and class III, class IIand class IV, and class I and class IV, or in the sandwich method usingthe antibodies in combination which did not recognize the regionsadjacent to each other. The present inventors further discovered thatPACAP38NH₂ was also detected in combinations of class I and class II,class II and class III, and class III and class IV, or in the sandwichmethod using the antibodies in combination which recognize the regionsadjacent to each other, and particularly that PACAP38NH₂ was detectedwith high sensitivity in the sandwich method in which the antibody ofclass II was used in combination with the antibody of class III. Thesandwich immunoassay is specific for PACAP38NH₂. For example, in thesandwich immunoassay using PA-6Na, one of the antibodies belonging toclass II, and PA-2Ca, one of the antibodies belonging to class III, itwas discovered that the cross reactivity with other peptides havinghomology with VIP and PACAP38NH₂ was 0.001% or less. Examples of suchpeptides include growth hormone releasing hormone (GRF) having thefollowing sequence (SEQ ID NO:9):

    ______________________________________                                        Tyr--Ala--Asp--Ala--Ile--Phe--Thr--Asn--Ser--Tyr--Arg--                       Lys--Val--Leu--Gly--Gln--Leu--Ser--Ala--Arg--Lys--Leu--                       Leu--Gln--Asp--Ile--Met--Ser--Arg--Gln--Gln--Gly--Glu--                       Ser--Asn--Gln--Glu--Arg--Gly--Ala--Arg--Ala--Arg--Leu--                       NH.sub.2,                                                                     ______________________________________                                    

and secretin, a typical gastrointestinal hormone having the followingsequence (SEQ ID NO:10)

    ______________________________________                                        His--Ser--Asp--Gly--Thr--Phe--Thr--Ser--Glu--Leu--Ser--                       Arg--Leu--Arg--Glu--Gly--Ala--Arg--Leu--Gln--Arg--                            Leu--Leu--Gln--Gly--Leu--Val--NH.sub.2                                        ______________________________________                                    

(molecular weight: 3039.4)

In the immunoassay for assaying PACAP27NH₂ by the sandwich method of thepresent invention, each of the antibodies used in the primary andsecondary reactions may be either the polyclonal antibody or themonoclonal antibody. It is however preferred that one of them is theantibody which recognizes the N-terminal portion of PACAP27NH₂ (classI), the region from the N-terminal portion to the central portion (classII) or C-terminal portion (class V) and the other is the antibody whichrecognizes regions other than the regions described above. Also in thissandwich immunoassay of the present invention, PACAP27NH₂ can bedetected without cross reaction with VIP, GRF or secretin (the crossreactivity is 0.001% or less). The sandwich immunoassay using either theantibody of class I or that of class II in combination with either theantibody of class III or that of class IV is specific for PACAP38NH₂ anddoes not cross react with PACAP27NH₂. On the other hand, the immunoassayfor assaying PACAP27NH₂ by the sandwich method using either the antibodyof class I or that of class II in combination with the antibody of classV shows a cross reactivity with PACAP38NH₂ as low as 0.22 to 3.6% byweight ratio or 0.31 to 5% by molar ratio. By using these immunoassays,therefore, PACAP38NH₂ and PACAP27NH₂ can be fractionated and determined.

The polyclonal antibody used in the present invention is usuallyprepared by producing a complex comprising a carrier protein and PACAPor a partial peptide of PACAP, which acts as an immunogen. Next, animalsare inoculated with this complex for immunization. The substancecontaining an anti-PACAP or anti-PACAP partial peptide antibody from theimmunized animals is recovered, and the antibody is then separated andpurified.

The monoclonal antibody of the present invention is prepared byselecting individuals having high antibody titer from theabove-mentioned immunized animals, recovering spleens or lymphaticcorpuscles therefrom 2 to 5 days after the final immunization, fusingantibody producing cells contained therein with myeloma cells, andselecting hybridoma cells which stably produce an antibody having hightiter to obtain monoclonal hybridoma cells.

Both of natural purified samples and synthetic samples can be used asimmunogens. PACAP and portions thereof are used. Compounds containingthe structure of PACAP or portions of PACAP are used as the immunogensin some cases.

The various peptides used in the present invention can be prepared bypeptide synthesis methods known to those skilled in the art. Either thesolid phase synthesis methods or the liquid phase synthesis methods maybe used. Examples of the peptide synthesis methods include methodsdescribed in B. Merrifield [J. Am. Chem. Soc. 85, 2149 (1963)], M.Bodanszky and M. A. Ondetti [Peptide Synthesis, Interscience Publishers,New York (1966)], Schroder and Lubke [The Peptide, Academic Press, NewYork (1965)], N. Izumiya et al. [Fundamentals and Experiments of PeptideSynthesis, Maruzen (1985)] and H. Yajima and S. Sakakibara [Course ofBiochemical Experiments 1, Chemistry of Proteins IV 205 (1977)].

For example, when PACAP38NH₂ or a partial peptide of PACAP38NH₂ issynthesized by the solid phase methods, using any of the insolubleresins known in the art such as chloromethyl resins,4-methylbenzhydrylamine resins and 4-oxymethylphenylacetamidomethylresins, protected amino acids are successively condensed to theC-terminal side of PACAP38NH₂ or of the partial peptide of PACAP38NH₂according to methods known in the art. Then, all protecting groups areremoved by hydrogen fluoride treatment, followed by purification bymethods known in the art such as high performance liquid chromatography,whereby the desired PACAP38NH₂ or partial peptide of PACAP38NH₂ can beobtained.

For example, the N-protected amino acids can be produced by protectingα-amino groups with Boc groups, the hydroxyl groups of serine andthreonine with Bzl groups, the ω-carboxylic acid groups of glutamic acidand aspartic acid with OBzl groups, the -amino group of lysine with aCl-Z group, the hydroxyl group of tyrosine with a Br-Z group, theguanido group of arginine with a Tos group, and the imidazole group ofhistidine with a Tos group.

The abbreviations used in this specification, are the abbreviationsadopted by IUPAC-IUB Commission on Biochemical Nomenclature or commonlyused in the art are employed. For example, the following abbreviationsare used. When an optical isomer is capable of existing with respect tothe amino acids, the L-form is represented unless otherwise specified.

PAM: Phenylacetamidomethyl

BHA: Benzhydrylamine

Boc: t-Butyloxycarbonyl

Cl-Z: 2-Chloro-benzyloxycarbonyl

Br-Z: 2-Bromo-benzyloxycarbonyl

Bzl: Benzyl

OBzl: Benzyl ester

Tos: p-Toluenesulfonyl

HOBt: 1-Benzotriazole

DCC: N,N'-Dichlorohexylcarbodiimide

Gly: Glycine

Ala: Alanine

Val: Valine

Leu: Leucine

Ile: Isoleucine

Ser: Serine

Thr: Threonine

Cys: Cysteine

Met: Methionine

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

His: Histidine

Phe: Phenylalanine

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gln: Glutamine

With respect to the protein complexes comprising the immunogens and thecarrier proteins used for immunization of mammals, any type of carrierproteins may be coupled with haptens in any ratio, as long as theantibodies can be produced effectively to the haptens coupled with thecarrier proteins to be immunized. For example, bovine serum albumin,bovine thyroglobulin or hemocyanin is coupled with the hapten in aweight ratio of 0.1 to 20, and preferably 1 to 5, per 1 of the hapten.

Various condensing agents may be used for the coupling of the haptensand the carrier proteins. In particular, glutaraldehyde, carbodiimideactive esters, maleimide active esters and active ester reagentscontaining thiol groups or dithiopyridyl groups are advantageously used.

The condensed products are given alone or with carriers or diluents towarm-blooded animals at sites where the antibodies are capable of beingproduced. In giving the condensed products, Freund's complete adjuvantor Freund's incomplete adjuvant may be given to enhance the antibodyproductivity. The condensed products are usually given once every 2 to 6weeks, 3 to 6 times in all.

The warm-blooded animals used therein include, for example, monkeys,rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens.

The antibodies are harvested from the blood, the ascites and the like(preferably from the blood) of the warm-blooded animals immunized by themethods described above. The titer of the anti-PACAP antibody in theantiserum is determined, for example, by reacting labeled PACAPdescribed below with the antiserum, and then measuring the activity of alabeling agent bound to the antibody. The antibodies are separated andpurified according to methods for separating and purifyingimmunoglobulin which are known in the art. Such methods include saltprecipitation, alcohol precipitation, isoelectric precipitation,electrophoresis, adsorption and desorption using an ion exchanger (forexample, DEAE), ultracentrifugation, gel filtration and specificpurifying methods for obtaining antibodies by recovering the antibodiesalone by using active adsorbents such as antigen-antibody complexes,protein A and protein G and then breaking the binding.

The antibodies thus obtained comprise mainly of IgG and also containadditional immunoglobulins such as IgM and IgA.

The anti-PACAP antibody producing hybridoma cells of the presentinvention can be prepared by selecting individuals having high antibodytiter from the warm-blooded animals such as mice immunized similarly tothe above method for preparing the polyclonal antibody, recoveringspleens or lymphatic corpuscles therefrom 2 to 5 days after the finalimmunization, and fusing antibody producing cells contained therein withmyeloma cells. The cells may be fused according to methods known in theart, for example, the method of Kohler and Milstein [Nature 256, 495(1975)]. Fusion accelerators include polyethylene glycol (PEG) andSendai virus. In particular, PEG is preferably used.

The myeloma cells include, for example, NS-1, P3Ul and SP2/0. Inparticular, P3Ul is preferably used. The ratio of the number of theantibody producing cells (splenic cells) to the myeloma cells ispreferably about 1:1 to 20:1. PEG(preferably PEG 1,000 to PEG 6,000) isadded in a concentration of about 10 to 80%, and incubated at 20° to 40°C., preferably 30° to 37° C. for 1 to 10 minutes, whereby the cellfusion can be effectively performed.

The anti-PACAP antibody producing hybridoma cells can be screened byvarious methods known in the art. Examples of such methods include anenzyme immunoassay (EIA) which comprises adding a hybridoma culturesupernatant to a solid phase (for example, a microplate) allowed toadsorb PACAP or the partial peptide thereof, then adding ananti-immunoglobulin antibody (when mouse cells are used for the cellfusion, an anti-mouse immunoglobulin antibody is employed) labeled withhorseradish peroxidase (HRP) or protein-A thereto, and detecting theanti-PACAP monoclonal antibody; and an enzyme immunoassay (EIA) whichcomprises adding a hybridoma culture supernatant to a solid phaseallowed to adsorb an anti-immunoglobulin antibody or protein A, thenadding HRP-labeled PACAP, and detecting the anti-PACAP monoclonalantibody bound to the solid phase. Selection and breeding of theanti-PACAP monoclonal antibodies are usually achieved by addition of HAT(hypoxanthine, aminopterin and thymidine) and by use of a medium foranimal cells containing 10 to 20% fetal calf serum, such as RPMI 1640.The antibody titer of the hybridoma culture supernatant can be assayedsimilarly to the above-mentioned method for assaying the titer of theanti-PACAP antibody in antiserum.

Isolation and purification of the anti-PACAP monoclonal antibodies areconducted in accordance with methods for isolating and purifyingimmunoglobulin, similar to the isolation and purification of thepolyclonal antibodies described above.

The anti-PACAP polyclonal antibody reactive to a partial region of PACAPcan be prepared by the above-mentioned method using a peptidecorresponding to the partial region thereof as a hapten forimmunization. Further, such anti-PACAP polyclonal antibody can also beprepared from the anti-PACAP polyclonal antibody prepared by using PACAPas a hapten, by use of affinity chromatography employeing a column towhich a peptide corresponding to the partial region thereof is bound.

Screening of hybridoma cells producing the anti-PACAP antibody reactiveto a partial region of PACAP and hybridoma cells producing theanti-PACAP monoclonal antibody reactive to PACAP, but not reactive tothe partial region thereof can be accomplished, for example, by assayingaffinity of the peptide corresponding to the above partial region forthe antibody produced by the hybridoma cells.

By using the anti-PACAP monoclonal and polyclonal antibodies obtainedabove, assay and tissue staining of PACAP can be carried out. For thesepurposes, antibody molecules themselves may be used, and F(ab')₂, Fab'or Fab fractions of the antibody molecules may be used.

PACAP is usually assayed by competitive methods which will be describedbelow. It is however preferable to use sandwich methods for the reasondescribed above.

In the competitive methods, the anti-PACAP antibody obtained in thepresent invention is competitively reacted with a test solution andlabeled PACAP, followed by measurement of the ratio of labeled PACAPbound to the antibody, thereby determining the amount of PACAP containedin the test solution.

The labeling agents for PACAP or for antibodies described below includeradioisotopes, enzymes, fluorescent substances and luminous substances.The radioisotopes include, for example, ¹²⁵ I, ¹³¹ I, ³ H and ¹⁴ C. Theenzymes which are stable and high in specific activity are preferablyused. Examples of such enzymes include β-galactosidase, β-gulcosidase,alkaline phosphatase, peroxidase and malate dehydrogenase. Thefluorescent substances include fluorescamine and fluoresceinisothiocyanate. The luminous substances include luminol, luminolderivatives, luciferin and lucigenin. Further, a biotin-avidin systemmay also be used in order to bind the labeling agent to the antibody orPACAP.

When the activity of the above-mentioned labeling agents is assayed, itis necessary to separate labeled PACAP bound to the antibody from freelabeled PACAP. This separation is hereinafter referred to as B/Fseparation for brevity. When the enzymes are used as the labelingagents, active adsorbents such as insolubilized antibodies to theanti-PACAP antibody or insolubilized protein A are advantageously usedas reagents for the B/F separation. For example, an anti-IgG antibody(corresponding to the antibody to the anti-PACAP antibody) is used asthe solid phase, and labeled PACAP binds to the anti-IgG antibody of thesolid phase through the above-mentioned antibody reactive thereto tomeasure the labeling agent on the solid phase. When the enzymes are usedas the labeling agents, the activity of the enzymes on an insolubilizedcarrier is assayed by ordinary colorimetric methods or fluorescentmethods. When the radioisotopes and the like are used as the labelingagents, reagents such as antibodies to the anti-PACAP antibody which arenot insolubilized, sodium sulfate, dextran charcoal powder andpolyethylene glycol are used for the B/F separation, in addition to theabove-mentioned reagents. In any methods, the activity of the labelingagent in the supernatant or in the precipitate is assayed.

The above-mentioned insolubilization may be achieved by physicaladsorption or chemical bonding usually used to insolubilize orimmobilize polysaccharides such as agarose, dextran and cellulose;synthetic resins such as polystyrene, polyacrylamide and silicone; andglass.

In the competitive methods, the anti-PACAP antibody, the test solution,labeled PACAP and the reagent for B/F separation can be reacted in anyorder. Also, all or a part of them may be reacted at the same time. Itis however preferable that at least labeled PACAP is added to thereaction system simultaneously with the reaction of the test solutionand the anti-PACAP antibody, or after the reaction. The reagents for theB/F separation such as sodium sulfate, dextran charcoal powder andpolyethylene glycol are mainly added to the reaction system at the finalstage thereof.

On the other hand, in the sandwich methods, the test solution is broughtinto contact with (or reacted with) the insolubilized anti-PACAPantibody (the primary reaction), and further the labeled anti-PACAPantibody is reacted therewith (the secondary reaction), followed byassay of the activity of the labeling agent on the insolubilizedcarrier, whereby the amount of PACAP in the test solution can bedetermined. The primary and secondary reactions may be conducted at thesame time or at different time. The labeling agents and theinsolubilizing methods can conform to those described above.

As the anti-PACAP antibody used in the secondary reaction, it ispreferred to use the antibody different from the anti-PACAP antibodyused in the primary reaction in the site to which PACAP binds.

Namely, the antibodies used in the primary and secondary reactions maybe polyclonal or monoclonal antibodies, respectively. For example,however, when the antibody used in the primary reaction recognizes theC-terminal portion of PACAP38NH₂ (class IV), it is preferred to use inthe secondary reaction the antibody which recognizes portions or regionsother than the C-terminal portion, namely recognizes the N-terminalportion (class I), the region from the N-terminal portion to the centralportion (class II) or the region from C-terminal portion to the centralportion (class III).

In the sandwich immunoassays, both of the antibody for solid phase andthe antibody for labeling may be antibodies of any class and subclass,and may be F(ab')₂, Fab' or Fab fractions which are obtained by removingFc' or Fc fractions therefrom, as long as they have antibody activity.

In the sandwich immunoassays, when the monoclonal antibody is used, itis not always necessary to use one kind of antibody as the antibody forsolid phase or for labeling. For the purpose of improving assayingsensitivity, mixtures of two or more kinds of antibodies can be used.

Further, the immunoassays using the antibodies which are obtainedaccording to the present invention can be used for diagnosis andtreatment of diseases related to PACAP.

Humors such as plasma, serum, urine, cerebrospinal fluid, ascites,pleural fluid and amniotic fluid, sputum and feces can be used as testsamples. These samples can be used for the immunoassays as such or withconcentration after dilution or extraction with various buffers.

Any buffers or organic solvents can be used as solvent for dilution orextraction of the samples. Preferred examples thereof include buffersfor immunoassay, water, physiological saline, acetate buffer, acetone,chloroform-methanol and these solutions containing surface activeagents. After extraction, the samples are sometimes heat treated. Thesamples may be concentrated directly under reduced pressure or underordinary pressure in a stream of nitrogen. Also, the samples may beadded to carriers for ion exchange or for reverse-phase chromatography,or to anti-PACAP antibody-bound carriers, and then eluted underappropriate conditions, followed by concentration under reduced pressureor under ordinary pressure in a stream of nitrogen. The carriers forreverse-phase chromatography includ C2, C8, C18 and phenyl cartridges.It is particularly preferable to use the anti-PACAP antibody-boundcarriers as the carriers for concentration. Condensates are dissolved inthe buffers for immunoassay, and then subjected to the immunoassays.

Further, the anti-PACAP antibodies obtained in the present invention canalso be used for immunohistochemical station of PACAP. Methods thereofcan be conducted, for example, in accordance with direct methods usingthe labeled anti-PACAP antibodies, and indirect methods using theanti-PACAP antibodies and the labeled antibodies to the anti-PACAPantibodies.

Furthermore, of the anti-PACAP antibodies obtained in the presentinvention, the antibody which can neutralize the adenylate cyclaseactivity of PACAP can be used as a specific neutralizing antibody.

As methods for screening an antibody specifically depressing theactivity of PACAP from the anti-PACAP antibodies, any methods fordetecting the parmacological activity of PACAP can be used. Examples ofsuch methods include an in vitro assay system in which measurements aremade based on the adenylate cyclase activity of PACAP in primary cultureof hypophyses or in culture systems of various cells containing browncytoma cell strain PC12h, and an in vivo assay system in whichmeasurements are made based on the vasodepressor activity of PACAP tothe experimental animals.

The antibodies specifically depressing the activity of PACAP may beantibodies of any class, such as IgG, IgA and IgM, and may be Fab' ofFab fractions which are obtained by removing Fc' or FC regionstherefrom, or polymers of the fractions. A chimera antibody can also beused which is obtained by fusing a variable gene region of a monoclonalantibody being specifically capable of depressing the activity of PACAPwith a constant gene region of human immunoglobulin, followed byexpression as a recombinant.

Hybridoma cells obtained in Example 8 described below were depositedwith the Institute for Fermentation, Osaka, Japan (IFO) on Feb. 27,1990, and with the Fermentation Research Institute, the Agency ofIndustrial Science and Technology, the Ministry of International Tradeand Industry, Japan (FRI) 1-3, Higashi-chome, Tsukuba-shi, Ibaraki-Kea305 Japan on March 16, 1991, under the following accession numbers.

    ______________________________________                                        Hybridoma cell IFO     FERM-BP (FRI)                                          ______________________________________                                        PA-1N          50225   2811                                                   PA-3N          50226   2812                                                   PA-5N          50227   2813                                                   PA-6N          50228   2814                                                   PA-2C          50229   2815                                                   PA-1C          50230   2816                                                   ______________________________________                                    

In the following Examples, antibodies obtained from the respectivehybridoma cells are represented by giving a symbol "a" after the namesof the cells.

REFERENCE EXAMPLE 1

Synthesis of PACAP38 NH₂

PACAP38 NH₂ was synthesized by using 1.04 g (0.5 mmole) of acommercially available p-methyl BHA resin (Applied Biosystems Inc.) anda peptide synthesizer (Model 430A, Applied Biosystems Inc.).

A starting amino acid, Boc-Lys(Cl-Z), was activated with HOBt/DCC andthen condensed to the resin. Thereafter, the Boc group on the resin wastreated with 50% trifluoroacetic acid/methylene chloride to deprotectthe amino group. To this free amino group, the following protected aminoacids activated with HOBt/DCC were condensed in turn according to theamino acid sequence of PACAP38:

    Boc-Asn, Boc-Lys(C1-Z), Boc-Val, Boc-Arg(Tos), Boc-Gln, Boc-Tyr(Br-Z), Boc-Gly, Boc-Leu, Boc-Ala, Boc-Met, Boc-Ser(Bzl), Boc-Asp(OBzl), Boc-Thr(Bzl), Boc-Phe, Boc-Ile, and Boc-His(Tos)

After the completion of each reaction, the residual amino groups wereacetylated with acetic anhydride to obtain 2.42 g of a protected PACAP38NH₂ resin.

0.51 g of the resulting protected PACAP38 NH₂ resin was treated with 5ml of hydrogen fluoride in the presence of 0.6 g of p-cresol at 0° C.for 60 minutes, followed by removal of excess hydrogen fluoride bydistillation under reduced pressure. The residue was washed twice with 5ml of ethyl ether, and then extracted with 6 ml of 50% aqueous aceticacid. The insoluble material was removed by filtration and washed with 5ml of 50% aqueous acetic acid. The filtrate and the washings werecombined, and the combined solution was concentrated to 2 to 3 ml. Theconcentrated solution was applied on a Sephadex LH-20 column (2×90 cm)for elution with 50% acetic acid. The main fractions were collected,followed by removal by distillation under reduced pressure. Then, theresidue was dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid.The resulting solution was subjected to a YMC-ODS AM120 S-50 resincolumn (1.6×7 cm) and eluted by a linear gradient of 0.1% aqueoustrifluoroacetic acid and 50% acetonitrile containing 0.1%trifluoroacetic acid.

The main fractions were combined, followed by lyophilization. Thus, 60mg of white powder was obtained. This powder was dissolved in 20 ml of0.05M aqueous ammonium acetate. The resulting solution was subjected toa CM-Cellulofine resin column (1×6 cm) and eluted by a linear gradientof from 0.05M to 1M ammonium acetate. The main fractions were combined.The combined solution was subjected to a YMC-ODS column (2.6×7 cm) againand eluted by a linear gradient of from 0% to 40% aqueous acetonitrilecontaining 0.1% trifluoroacetic acid. The fractions of 28% to 30%acetonitrile were collected, followed by lyophilization. Thus, 21.6 mgof white powder was obtained.

Anal. for amino acids: Asp 2.90(3), Thr 0.84(1), Ser 2.10(3), Glu2.21(2), Gly 2.00(2), Ala 3.29(3), Val 3.19(3), Met 1.01(1), Ile0.87(1), Leu 2.19(2), Tyr 3.93(4), Phe 0.92(1), Lys 7.18(7), His0.96(1), Arg 4.19(4) (M+H)⁺ by mass spectrography (SIMS): 4530 HPLCelution time: 19.6 minutes Column conditions Column: YMC-ODS (AM-301,S-5 120A) Eluent: A (0.1% aqueous trifluoroacetic acid) B (acetonitrilecontaining 0.1% trifluoroacetic acid) A linear gradient elution from theeluent A to the eluent B for 50 minutes Flow rate: 1.0 ml/minute

REFERENCE EXAMPLE 2

Synthesis of PACAP27 NH₂

PACAP27 NH₂ was synthesized by using 1.04 g (0.5 mmole) of acommercially available p-methyl BHA resin (Applied Biosystems Inc.) anda peptide synthesizer (Model 430A, Applied Biosystems Inc.).

A starting amino acid, Boc-Leu, was activated with HOBt/DCC and thencondensed to the resin. Thereafter, the Boc group on the resin wastreated with 50% trifluoroacetic acid/methylene chloride to deprotectthe amino group. To this free amino group, the following protected aminoacids activated with HOBt/DCC were condensed in turn according to theamino acid sequence of PACAP38 (1-27):

    Boc-Val, Boc-Ala, Boc-Leu, Boc-Tyr(Br-Z), Boc-Lys(Cl-Z), Boc-Met, Boc-Gln, Boc-Arg(Tos), Boc-Ser(Bzl), Boc-Asp(OBzl), Boc-Thr(Bzl), Boc-Phe, Boc-Ile, and Boc-His(Tos)

After the completion of each reaction, the residual amino groups wereacetylated with acetic anhydride to obtain 2.31 g of a protected PACAP27NH₂ resin.

0.79 g of the resulting protected PACAP27 NH₂ resin was treated with 10ml of absolute hydrogen fluoride in the presence of 1.2 g of p-cresol at0° C. for 60 minutes, followed by removal of excess hydrogen fluoride bydistillation under reduced pressure. The residue was washed twice with 5ml of ethyl ether, and then extracted with 5 ml of 50% aqueous aceticacid. The insoluble material was removed by filtration and washed with 5ml of 50% aqueous acetic acid. The filtrate and the washings werecombined, and the combined solution was concentrated to 2 to 3 ml. Theconcentrated solution was applied on a Sephadex LH-20 column (2×75 cm)for elution with 50% acetic acid. The main fractions were collected,followed by distillation under reduced pressure. The residue wasdissolved in 100 ml of 0.1% aqueous trifluoroacetic acid. The resultingsolution was subjected to a YMC-ODS AM120 S-50 resin column (2.6×7 cm)and eluted by a liner gradient of 0.1% aqueous trifluoroacetic acid and50% acetonitrile containing 0.1% trifluoroacetic acid. The mainfractions were combined and the combined solution was applied onto aYMC-ODS column (2.6×7 cm) again and eluted by a linear concentrationgradient with from 15 to 35% aqueous acetonitrile solution containing0.1% trifluoroactetic acid. The acetonitrile 30 to 32% fractions werecollected, followed by lyophilization. The resulting product wasdissolved in 20 ml of 0.05M-aqueous ammonium acetate. The solution wasapplied onto a CM--Cellulofine resin column (1×6 cm) and eluted by alinear concentration gradient with water to 0.33M--aqueous ammoniumacetate.

The main fractions (0.18 to 0.22M) were collected, followed bylyophilization. Thus, 20 mg of white powder was obtained.

Anal. for amino acids: Asp 1.96(2), Thr 0.94(1), Ser 2.57(3), Glu1.07(1), Gly 0.95(1), Ala 3.00(3), Val 1.96(2), Met 0.88(1), Ile0.88(1), Leu 1.93(2), Tyr 2.87(3), Phe 0.90(1), Lys 2.91(3), His0.94(1), Arg 2.17(2) (M+H)⁺ by mass spectrography (SIMS): 3146.7 HPLSelution time: 21.2 minutes Column conditions Column: YMC-ODS (AM-301,S-5 120A) Eluent: A (0.1% aqueous trifluoroacetic acid) B (acetonitrilecontaining 0.1% trifluoroacetic acid) A linear gradient elution from theeluent A to the eluent B for 50 minutes Flow rate: 1.0 ml/minute

REFERENCE EXAMPLE 3

Synthesis of PACAP27 OH (His ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser ArgTyr Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu-OH) (aminoacid numbers 1-27 of SEQ ID NO:1).

PACAP27 OH was synthesized by using 0.60 g (0.5 mmole) of a commerciallyavailable Boc-Leu-OCH₂ -PAM resin (Applied Biosystems Inc.) and apeptide synthesizer (Model 430A, Applied Biosystems Inc.).

The Boc group on the resin was treated with 50% trifluoroaceticacid/methylene chloride to deprotect the amino group. To this free aminogroup, the following protected amino acids activated with HOBt/DCC werecondensed in turn according to the amino acid sequence of PACAP27:

    Boc-Val, Boc-Lys(C1-Z), Boc-Arg(Tos), Boc-Gln, Boc-Tyr(Br-Z), Boc-Gly, Boc-Leu, Boc-Ala, Boc-Met, Boc-Ser(Bzl), Boc-Asp(OBzl), Boc-Thr(Bzl), Boc-Phe, Boc-Ile, and Boc-His(Tos)

After the additional condensation by the amino acids activated by DCC orHOBt/DCC, the unreacted amino groups were acetylated with aceticanhydride to obtain 1.25 g of a protected PACAP27 OH resin.

0.65 g of the resulting protected PACAP27-OCH₂ -PAM resin was treatedwith 6 ml of absolute hydrogen fluoride in the presence of 1.0 g ofp-cresol at 0° C. for 60 minutes, followed by removal of excess hydrogenfluoride by distillation under reduced pressure. The residue was washedtwice with 5 ml of ethyl ether, and then extracted with 5 ml of 50%aqueous acetic acid. The insoluble material was removed by filtrationand washed with 5 ml of 50% aqueous acetic acid. The filtrate and thewashings were combined, and the combined solution was concentrated to 2to 3 ml under reduced pressure. The concentrated solution was applied ona Sephadex LH-20 column (2×75 cm) for elution with 50% acetic acid. Themain fractions were collected, followed by distillation under reducedpressure. The residue was dissolved in 100 ml of 0.1% aqueoustrifluoroacetic acid. The resulting solution was subjected to a YMC-ODSAM120 S-50 resin column (2.6×7 cm) and eluted by a liner gradient of0.1% aqueous trifluoroacetic acid and 50% acetonitrile containing 0.1%trifluoroacetic acid. The main fractions were combined and the combinedsolution was applied onto a YMC-ODS column (2.6×7 cm) again and elutedby a linear concentration gradient with from 15 to 40% aqueousacetonitrile solution containing 0.1% trifluoroactetic acid. Theacetonitrile 25 to 28% fractions were collected, followed bylyophilization. The resulting product was dissolved in 20 ml of0.05M-aqueous ammonium acetate. The solution was applied onto aCM--Cellulofine resin column (1×6 cm) and eluted by a linearconcentration gradient with 0.05M to 0.33M--aqueous ammonium acetate.

The main fractions were collected, followed by lyophilization. Thus, 20mg of white powder was obtained.

Anal. for amino acids: Asp 2.03(2), Thr 0.96(1), Ser 2.66(3), Glu1.08(1), Gly 1.01(1), Ala 305(3), Val 1.98(2), Met 0.94(1), Ile 0.94(1),Leu 2.00(2), Tyr 2.96(3), Phe 0.95,(1), Lys 2.99(3), His 1.03(1), Arg2.25(2) (M+H)⁺ by mass spectrography (SIMS): 3147.9 HPLS elution time:18.69 minutes Column conditions Column: YMC-ODS (AM-301, S-5 120A)(4.6×100) Eluent: A (0.1% aqueous trifluoroacetic acid) B (acetonitrilecontaining 0.1% trifluoroacetic acid) A linear gradient elution from theeluent A to the eluent B for 25 minutes Flow rate: 1.0 ml/minute

EXAMPLE 1-1

Synthesis of PACAP(14-38) NH₂

PACAP(14-38) NH₂ was synthesized by using 1.04 g (0.5 mmole) of acommercially available p-methyl BHA resin (Applied Biosystems Inc.) anda peptide synthesizer (Model 430A, Applied Biosystems Inc.).

A starting amino acid, Lys(Cl-Z), was activated with HOBt/DCC and thencondensed to the resin. Thereafter, the Boc group on the resin wastreated with 50% trifluoroacetic acid/methylene chloride to deprotectthe amino group. To this free amino group, the following protected aminoacids activated with HOBt/DCC were condensed in turn according to theamino acid sequence of PACAP(14-38) NH₂ :

    Boc-Asn, Boc-Lys(C1-Z), Boc-Val, Boc-Arg(Tos), Boc-Gln, Boc-Tyr(Br-Z), Boc-Gly, Boc-Leu, Boc-Ala, Boc-Met

After the additional condensation by the amino acids activated by DCC orHOBt/DCC, the unreacted amino groups were acetylated with aceticanhydride to obtain 2.00 g of a protected PACAP(14-38) NH₂ resin.

0.48 g of the resulting protected PACAP(14-38) NH₂ resin was treatedwith 5 ml of absolute hydrogen fluoride in the presence of 0.48 g ofp-cresol at 0° C. for 60 minutes, followed by removal of excess hydrogenfluoride by distillation under reduced pressure. The residue was washedtwice with 5 ml of ethyl ether, and then extracted with 5 ml of 50%aqueous acetic acid. The insoluble material was removed by filtrationand washed with 5 ml of 50% aqueous acetic acid. The filtrate and thewashings were combined, and the combined solution was concentrated to 2to 3 ml. The concentrated solution was applied on a Sephadex LH-20column (2×75 cm) for elution with 50% acetic acid. The main fractionswere collected, followed by distillation under reduced pressure. Theresidue was dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid.The resulting solution was subjected to a YMC-ODS AM120 S-50 resincolumn (2.6×7 cm) and eluted by a liner gradient of 0.1% aqueoustrifluoroacetic acid and 30% acetonitrile containing 0.1%trifluoroacetic acid. The main fractions were collected, followed bylyophilization. Thus, 20.2 mg of white powder was obtained.

Anal. for amino acids: Asp 1.01(1), Glu 2.01(2), Gly 1.00(1), Ala3.01(3), Val 2.85(3), Met 0.86(1), Leu 2.08(2), Tyr 1.98(2), Lys6.37(7), Arg 3.24(3) (M+H)⁺ by mass spectrography (SIMS): 3003.6 HPLSelution time: 13.1 minutes Column conditions Column: YMC-ODS (AM-301,S-5 120A) Eluent: A (0.1% aqueous trifluoroacetic acid) B (acetonitrilecontaining 0.1% trifluoroacetic acid) A linear gradient elution from theeluent A to the eluent B for 25 minutes Flow rate: 1.0 ml/minute

EXAMPLE 1-2

Synthesis of PACAP(1-13) OH

PACAP(1-13) OH was synthesized by using 0.87 g (0.5 mmole) of acommercially available Boc-Tyr(Br-Z)-OCH₂ -PAM resin (Applied BiosystemsInc.) and a peptide synthesizer (Model 430A, Applied Biosystems Inc.).

The Boc group on the resin was treated with 50% trifluoroaceticacid/methylene chloride to deprotect the amino group. To this free aminogroup, the following protected amino acids activated with HOBt/DCC werecondensed in turn according to the amino acid sequence of PACAP(1-13):

    Boc-Arg(Tos), Boc-Tyr(Br-Z), Boc-Gly, Boc-Ser(Bzl), Boc-Asp(OBzl), Boc-Thr(Bzl), Boc-Phe, Boc-Ile, and Boc-His(Tos)

After the additional condensation by the amino acids activated by DCC orHOBt/DCC, the unreacted amino groups were acetylated with aceticanhydride to obtain 1.86 g of a protected PACAP(1-13)OCH₂ -PAM resin.

0.70 g of the resulting protected resin was treated with 10 ml ofabsolute hydrogen fluoride in the presence of 0.81 g of p-cresol at 0°C. for 60 minutes, followed by removal of excess hydrogen fluoride bydistillation under reduced pressure. The residue was washed twice with 5ml of ethyl ether, and then extracted with 5 ml of 50% aqueous aceticacid. The insoluble material was removed by filtration and washed with 5ml of 50% aqueous acetic acid. The filtrate and the washings werecombined, and the combined solution was concentrated to 2 to 3 ml underreduced pressure. The concentrated solution was applied on a SephadexLH-20 column (2×75 cm) for elution with 50% acetic acid. The mainfractions were collected, followed by distillation under reducedpressure. The residue was dissolved in 100 ml of 0.1% aqueoustrifluoroacetic acid. The resulting solution was subjected to a YMC-ODSAM120 S-50 resin column (2.6×7 cm) and eluted by a liner gradient of0.1% aqueous trifluoroacetic acid and 33% acetonitrile containing 0.1%trifluoroacetic acid. The main fractions were combined and the combinedsolution was purified again under the same column conditions. The mainfractions were collected, followed by lyophilization. Thus, 38 mg ofwhite powder was obtained.

Anal. for amino acids: Asp 2.00(2) , Thr 0.93(1) , Ser 2.43(3) , Glu1.05(1) , Gly 1.00(1), Tyr 1.82(2), Phe 1.02(1), His 1.31(1), Arg1.12(1) (M+H)⁺ by mass spectrography (SIMS): 1547.5 HPLS elution time:12.3 minutes Column conditions Column: YMC-ODS (AM-301, S-5 120A)Eluent: A (0.1% aqueous trifluoroacetic acid) B (acetonitrile containing0.1% trifluoroacetic acid) A linear gradient elution from the eluent Ato the eluent B for 25 minutes Flow rate: 1.0 ml/minute

EXAMPLE 1-3

Synthesis of PACAP(4-27) OH

PACAP(4-27) OH was synthesized by using 0.60 g (0.5 mmole) of acommercially available Boc-Leu-OCH₂ -PAM resin (Applied Biosystems Inc.)and a peptide synthesizer (Model 430A, Applied Biosystems Inc.).

The Boc group on the resin was treated with 50% trifluoroaceticacid/methylene chloride to deprotect the amino group. To this free aminogroup, the following protected amino acids activated with HOBt/DCC werecondensed in turn according to the amino acid sequence of PACAP(4-27):

    Boc-Lys(Cl-Z), Boc-Val, Boc-Arg(Tos), Boc-Gln, Boc-Tyr(Br-Z), Boc-Gly, Boc-Leu, Boc-Ala, Boc-Met, Boc-Ser(Bzl), Boc-Asp(OBzl), Boc-Thr(Bzl), Boc-Phe, Boc-Ile

After the additional condensation by the amino acids activated by DCC orHOBt/DCC, the unreacted amino groups were acetylated with aceticanhydride to obtain 1.08 g of a protected PACAP(4-27)OCH₂ -PAM resin.

0.29 g of the resulting protected resin was treated with 5 ml ofabsolute hydrogen fluoride in the presence of 0.49 g of p-cresol at 0°C. for 60 minutes, followed by removal of excess hydrogen fluoride bydistillation under reduced pressure. The residue was washed twice with 5ml of ethyl ether, and then extracted with 5 ml of 50% aqueous aceticacid. The insoluble material was removed by filtration and washed with 5ml of 50% aqueous acetic acid. The filtrate and the washings werecombined, and the combined solution was concentrated to 2 to 3 ml underreduced pressure. The concentrated solution was applied on a SephadexLH-20 column (2×75 cm) for elution with 50% acetic acid. The mainfractions were collected, followed by distillation under reducedpressure. The residue was dissolved in 100 ml of 0.1% aqueoustrifluoroacetic acid. The resulting solution was subjected to a YMC-ODSAM120 S-50 resin column (2.6×7 cm) and eluted by a liner gradient of 15%acetonitrile containing 0.1% trifluoroacetic acid and 50% acetonitrilecontaining 0.1% trifluoroacetic acid. The main fractions were collected,followed by lyophilization to obtain 33 mg of white powder. The powderwas dissolved in 20 ml of 0.05M-aqueous ammonium acetate. The solutionwas applied onto a CM-Cellurofine resin column (1×6 cm) and eluted by alinear gradient with water to 0.30M-aqueous ammonium acetate. The mainfractions (0.18 to 0.22M) were collected, followed by lyophilization.Thus, 33 mg of white powder was obtained.

Anal. for amino acids: Asp 1.02(1) , Thr 0.98(1) , Ser 1.78(3) , Glu1.07(1) , Gly 1.02(1) , Ala 3.04(3) , Val 1.89(2) , Met 0.81(1) , Ile0.89(1), Leu 2.00(2), Tyr 2.91(3), Phe 0.90(1), Lys 2.89(3), Arg 2.20(2)(M+H)⁺ by mass spectrography (SIMS): 2808.5 HPLS elution time: 14.5minutes Column conditions Column: YMC-ODS (AM-301, S-5 120A) Eluent: A(0.1% aqueous trifluoroacetic acid) B (acetonitrile containing 0.1%trifluoroacetic acid) A linear gradient elution from the eluent A to theeluent B for 35 minutes Flow rate: 1.0 ml/minute

EXAMPLE 1-4

Synthesis of PACAP(31-38) NH₂

PACAP(31-38) NH₂ was synthesized by using 0.98 g (0.5 mmole) of acommercially available p-methyl BHA resin (Applied Biosystems Inc.) anda peptide synthesizer (Model 430A, Applied Biosystems Inc.).

A starting amino acid, Boc-Lys(Cl-Z), was activated with HOBt/DCC andthen condensed to the resin. Thereafter, the Boc group on the resin wastreated with 50% trifluoroacetic acid/methylene chloride to deprotectthe amino group. To this free amino group, the following protected aminoacids activated with HOBt/DCC were condensed in turn according to theamino acid sequence of PACAP(14-38) NH₂ :

    Boc-Asn, Boc-Lys(C1-Z), Boc-Val, Boc-Arg(Tos), Boc-Gln, Boc-Tyr(Br-Z)

After the additional condensation by the amino acids activated by DCC orHOBt/DCC, the unreacted amino groups were acetylated with aceticanhydride to obtain 2.00 g of a protected PACAP(31-38) NH₂ resin.

0.43 g of the resulting protected PACAP(31-38) NH₂ resin was treatedwith 5 ml of absolute hydrogen fluoride in the presence of 0.6 g ofp-cresol at 0° C. for 60 minutes, followed by removal of excess hydrogenfluoride by distillation under reduced pressure. The residue was washedtwice with 5 ml of ethyl ether, and then extracted with 5 ml of 50%aqueous acetic acid. The insoluble material was removed by filtrationand washed with 5 ml of 50% aqueous acetic acid. The filtrate and thewashings were combined, and the combined solution was concentrated to 2to 3 ml. The concentrated solution was applied on a Sephadex LH-20column (2×75 cm) for elution with 50% acetic acid. The main fractionswere collected, followed by distillation under reduced pressure. Theresidue was dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid.The resulting solution was subjected to a YMC-ODS AM120 S-50 resinColumn (2.6×7 cm) and eluted by a liner gradient of 0.1% aqueoustrifluoroacetic acid and 33% acetonitrile containing 0.1%trifluoroacetic acid. The main fractions were collected, followed bylyophilization. Thus, 45 mg of white powder was obtained.

Anal. for amino acids: Asp 1.02(1), Glu 1.05(1), Val 1.00(1), Tyr0.90(1), Lys 2.98(3), Arg 1.12(1) (M+H)⁺ by mass spectrography (SIMS):1062.7 HPLS elution time: 11.6 minutes Column conditions Column: YMC-ODS(AM-301, S-5 120A) Eluent: A (0.1% aqueous trifluoroacetic acid) B(acetonitrile containing 0.1% trifluoroacetic acid) A linear gradientelution from the eluent A to the eluent mixture [A:B(4:1)] for 20minutes Flow rate: 1.0 ml/minute

EXAMPLE 1-5

Synthesis of [Cys¹⁰ ]PACAP(11-27) NH₂

[Cys¹⁰ ]PACAP(11-27) NH₂ was synthesized by using 0.66 g (0.5 mmole) ofa commercially available p-methyl BHA resin (Applied Biosystems Inc.)and a peptide synthesizer (Model 430A, Applied Biosystems Inc.).

A starting amino acid, Boc-Leu, was activated with HOBt/DCC and thencondensed to the resin. Thereafter, the Boc group on the resin wastreated with 50% trifluoroacetic acid/methylene chloride to deprotectthe amino group. To this free amino group, the following protected aminoacids activated with HOBt/DCC were condensed in turn according to theamino acid sequence of [Cys¹⁰ ]PACAP(11-27) NH₂ :

    Boc-Val, Boc-Ala, Boc-Tyr(Br-Z), Boc-Lys(Cl-Z), Boc-Met, Boc-Gln, Boc-Arg(Tos), Boc-Ser(Bzl), Boc-Cys(MeBzl)

After the additional condensation by the amino acids activated by DCC orHOBt/DCC, the unreacted amino groups were acetylated with aceticanhydride to obtain 1.20 g of a protected [Cys¹⁰ ]PACAP(11-27) NH₂resin.

0.60 g of the resulting protected resin was treated with 10 ml ofabsolute hydrogen fluoride in the presence of 1.0 g of p-cresol at 0° C.for 60 minutes, followed by removal of excess hydrogen fluoride bydistillation under reduced pressure. The residue was washed twice with 5ml of ethyl ether, and then extracted with 5 ml of 50% aqueous aceticacid. The insoluble material was removed by filtration and washed with 5ml of 50% aqueous acetic acid. The filtrate and the washings werecombined, and the combined solution was concentrated to 2 to 3 ml. Theconcentrated solution was applied on a Sephadex LH-20 column (2×75 cm)for elution with 50% acetic acid. The main fractions were collected,followed by distillation under reduced pressure. The residue wasdissolved in 100 ml of 0.1% aqueous trifluoroacetic acid. The resultingsolution was subjected to a YMC-ODS AM120 S-50 resin column (2.6×7 cm)and eluted by a liner gradient of 0.1% aqueous trifluoroacetic acid and50% acetonitrile containing 0.1% trifluoroacetic acid. The mainfractions were collected, followed by lyophilization. Thus, 70 mg ofwhite powder was obtained.

Anal. for amino acids: Ser 0.92(1), Glu 1.07(1), Ala 2.00(2), Val1.96(2), Met 0.88(1), Leu 1.93(2), Tyr 1.87(2), Lys 2.91(3), Arg 2.17(2)(M+H)⁺ by mass spectrography (SIMS): 2127.9 HPLS elution time: 20.8minutes Column conditions Column: YMC-ODS (AM-301, S-5 120A) Eluent: A(0.1% aqueous trifluoroacetic acid) B (acetonitrile containing 0.1%trifluoroacetic acid) A linear gradient elution from the eluent A to theeluent B for 50 minutes Flow rate: 1.0 ml/minute

EXAMPLE 2-1 Preparation of Immunogen Containing PACAP38NH₂

A complex comprising PACAP38NH₂ obtained in Reference Example 1described above and bovine thyroglobulin (hereinafter referred to asBTG) was prepared, and it was used as an immunogen.

Namely, 2.8 mg of PACAP38NH₂ and 8.4 mg of BTG were dissolved in 1 ml of0.1M phosphate buffer (pH 6.9), and glutaraldehyde was added thereto toa final concentration of 0.04%, followed by reaction at room temperaturefor 2 hours. After reaction, the resulting product was dialyzed againstphysiological saline at 4° C. for 2 days.

EXAMPLE 2-2 Preparation of Immunogen Containing PACAP(11-27)NH₂

A complex comprising [Cys¹⁰ ]PACAP(11-27)NH₂ obtained in Example 1-5described above and BTG was prepared, and it was used as an immunogen.

Namely, 20 mg of BTG was dissolved in 1.4 ml of 0.1M phosphate buffer(pH 6.9). The resulting solution was mixed with 100 μl of a DMF solutioncontaining 2.2 mg (8 μmoles) of N-(γ-maleimidobutyryloxy)succinimide(hereinafter referred to as GMBS), followed by reaction at roomtemperature for 40 minutes. After reaction, the resulting product wasfractionated on a Sephadex G-25 column, thereby obtaining a maleimidegroup-introduced BTG. Then, 12 mg of the maleimide group introduced BTGand 3.0 mg of [Cys¹⁰ ]PACAP(11-27)NH₂ were mixed and reacted with eachother at 4° C. for 3 days. After reaction, the reaction product wasdialyzed against physiological saline at 4° C. for 2 days.

EXAMPLE 3-1 Immunization of PACAP38NH₂ -BTG Conjugate

The female mice BALB/C 6 to 8 weeks old were subcutaneously immunizedwith 80 μg/mouse of the immunogen PACAP38NH₂ -BTG complex, obtained inExample 2-1 described above, together with Freund's complete adjuvant.Then, the mice were additionally immunized with the same amount of theimmunogen, together with Freund's incomplete adjuvant, 2 to 3 times at4-week intervals.

EXAMPLE 3-2 Immunization of PACAP(11-27)NH₂ -BTG Conjugate

The male rabbits were subcutaneously immunized with 400 μg/rabbit of thePACAP(11-27)NH₂ -BTG complex, obtained in Example 2-2 described above,together with Freund's complete adjuvant. Then, the rabbits wereadditionally immunized with the same amount of the immunogen, togetherwith Freund's incomplete adjuvant, 6 times at 4-week intervals.

EXAMPLE 4-1 Preparation of Horseradish Peroxidase (HRP)-LabeledPACAP38NH₂

A marker for the enzyme immunoassay (EIA) was prepared by crosslinkingPACAP38NH₂ obtained in Reference Example 1 and HRP.

Namely, 180 nmoles of PACAP38NH₂ was dissolved in 500 μl of 0.1Mphosphate buffer (pH 6.8), and 50 μl of a DMF solution containing 450nmoles of GMBS was mixed therewith, followed by reaction at roomtemperature for 30 minutes. After reaction, the resulting product wasfractionated on a Sephadex G-15 column. Thus, 100 nmoles of a maleimidegroup-introduced polypeptide was obtained.

On the other hand, 7.9 mg (200 nmoles) of HRP was dissolved in 0.95 mlof 0.02M phosphate buffer (pH 6.8) containing 0.15M NaCl, and 50 μl of aDMF solution containing 1.54 mg (4.9 μmoles) ofN-succinimidyl-3-(2-pyridylthio)propionate (hereinafter referred asSPDP) was mixed therewith, followed by reaction at room temperature for40 minutes. After reaction, 0.33 ml of 0.1M acetate buffer (pH 4.5)containing 8.2 mg (53 μmoles) of dithiothreitol was added thereto,followed by reaction at room temperature for 20 minutes. Then, thereaction product was fractionated on a Sephadex G-25 column. Thus, 6 mg(100 nmoles) of a SH group-introduced enzyme was obtained.

Then, 100 nmoles of maleimide group-introduced PACAP38NH₂ and 100 nmolesof SH group-introduced HRP were mixed and reacted with each other at 4°C. for 16 hours. After reaction, the reaction product was fractionatedon an Ultrogel AcA44 (LKB-Pharmacia) to obtain HRP-labeled PACAP38NH₂.

EXAMPLE 4-2 Preparation of HRP-Labeled PACAP(11-27)NH₂

In 950 μl of 180 nmoles of 0.1M phosphate buffer (pH 6.8) was dissolved8 mg (200 nmoles) of HRP, and 50 μl of a DMF solution containing 1.4 mg(5 μnmoles) of GMBS was mixed therewith, followed by reaction at roomtemperature for 40 minutes. Thereafter, the resulting product wasfractionated on a Sephadex G-15 column to obtain maleimidegroup-introduced HRP. Then, 3.3 mg (78 nmoles) of maleimidegroup-introduced HRP thus prepared and 0.65 mg (310 nmoles) of [Cys¹⁰]PACAP(11-27)NH₂ prepared in Example 1-5 were mixed and reacted witheach other at 4° C. for 1 day. After reaction, the reaction product wasfractionated on an Ultrogel AcA44 (LKB-Pharmacia) to obtain HRP-labeled[Cys¹⁰ ]PACAP(11-27)NH₂.

EXAMPLE 5-1

Determination of Antibody Titer of Mouse Antiserum

The antibody titer of the mouse antiserum was determined by thefollowing method. In order to prepare an anti-mouse immunoglobulinantibody-bound microplate, 100 μl of 0.1M carbonate buffer (pH 9.6)containing 100 μg/ml of the anti-mouse immunoglobulin antibody [IgGfraction, Kappel) was first poured into each well of a 96-wellmicroplate, and the plate was allowed to stand at 4° C. for 24 hours.After the plate was washed with phosphate buffered saline (hereinafterreferred to as PBS), 300 μl of PBS containing 25% Blockace (Snow BrandMilk Products) was poured into each well to block excess binding sitesof the wells, and treated at a temperature of at least 4° C. for 24hours.

To each well of the above-mentioned anti-mouse immunoglobulinantibody-bound microplate were added 50 μl of buffer E [0.02M phosphatebuffer (pH 7.0) containing 10% Blockace, 2 mg/ml bovine serum albumin(hereinafter referred to as BSA), 0.4M NaCl, 2 mM EDTA and 0.1% NaN₃ ]and 50 μl of the mouse anti-PACAP38NH₂ antiserum diluted with buffer E,followed by reaction at 4° C. for 16 hours. After the plate was washedwith PBS, 100 μl of the HRP-labeled PACAP38NH₂ prepared in Example 4-1described above [diluted 100 times with buffer H (pH 7.0) containing 2mg/ml BSA and 0.15M NaCl was added to each well, followed by reaction atroom temperature for 6 hours. After reaction, the plate was washed withPBS, and then 100 μl of 0.1M citrate buffer (pH 5.5) containing 0.2%o-phenylenediamine and 0.02% hydrogen peroxide was poured into each wellto assay the enzyme activity on the solid phase, followed by reaction atroom temperature for 10 minutes. After 100 μl of 4N sulfuric acid wasadded thereto to terminate the reaction, the absorption at 492 nm wasmeasured by a plate reader (MTP-32, Corona).

The results are shown in FIG. 1. Increases in anti-PACAP38 antibodytiter were observed in 4 mice of the 8 immunized mice.

EXAMPLE 5-2 Determination of Antibody Titer of Rabbit Antiserum

The antibody titer of the rabbit antiserum was determined in a similarmanner. An anti-rabbit immunoglobulin antibody (IgG fraction,Kappel)-bound microplate was prepared in the same manner as with Example5-1 described above. To each well of the above plate were added 50 μl ofbuffer E and 50 μl of the rabbit anti-PACAP(11-27)NH₂ antiserum dilutedwith buffer E, followed by reaction at 4° C. for 16 hours. After theplate was washed with PBS, 100 μl of HRP-labeled PACAP(11-27)NH₂prepared in Example 4-2 described above [diluted 200 times with buffer Hwas added to each well, followed by reaction at room temperature for 6hours. After reaction, the plate was washed with PBS, and then theenzyme activity on the solid phase was assayed by the use of the TMBmicrowell peroxidase substrate system (Kirkegaaed & Perry Lab., Inc.,sold by Funakoshi Yakuhin). A plate reader (MTP-32, Corona) was used formeasurement of the absorbance (at 450 nm).

The results are shown in FIG. 2. High antibody titer was detected in allof the immunized rabbits.

EXAMPLE 6 Cell Fusion

Mouse No. 5 which showed relatively high antibody titer was inoculatedwith a solution prepared by dissolving 200 μg of the immunogen in 0.25ml of physiological saline to conduct the final immunization. The spleenwas taken out of the mouse after 3 days from the final immunization,pressed by a stainless mesh, filtered, and floated in Eagle's minimumessential medium (MEM), thereby obtaining a spleen cell-floatingsolution. As a cell for cell fusion, BALB/C mouse-derived myeroma cellP3-X63.Ag8.Ul (P3Ul ) was used [Current Topics in Microbiology andImmunology 81, 1 (1978)]. The cell fusion was carried out in accordancewith the original method [Nature 256, 495 (1957)]. Namely, the spleencells and the P3Ul cells were washed 3 times with serum-free MEM, andmixed with each other so that the number ratio of the spleen cells tothe P3Ul cells reached 5:1. The mixture was centrifuged at 800 rpm for15 minutes to precipitate the cells. After the supernatant wasthoroughly removed, the precipitate was lightly loosened, and 0.3 ml of45% polyethylene glycol (PEG) 600 (Kochlight) was added thereto. Then,the mixture was allowed to stand in a hot water bath at 37° C. for 7minutes to perform the fusion. After the fusion was completed, MEM wasadded to the cells at a rate of 2 ml per minute. After 12 ml of MEM wasadded in total, the supernatant was removed by centrifugation at 600 rpmfor 15 minutes. The resulting precipitate was floated in GIT medium(GIT-10FCS, Wako Pure Chemical Industries) containing 10% fetal calfserum so that the P3Ul cells were contained in an amount of 2×10⁶cells/ml. The resulting suspension was seeded in 120 wells of a 24-wellmulti-dish (Linbro) in an amount of 1 ml/well. After seeding, the cellswere incubated in a 5% carbon dioxide incubator at 37° C. After 24hours, 1 ml of GIT-10FCS medium (HAT medium) containing HAT (1×10⁻⁴ Mhypoxanthine, 4×10⁻⁷ M aminopterin and 1.6×10⁻³ M thymidine) was addedto each well to initiate HAT selective culture. The HAT selectiveculture was continued by discarding 1 ml of old liquor and thensupplying 1 ml of HAT medium, 3, 6 and 9 days after the initiation ofthe culture. The proliferation of hybridoma cells was observed 9 to 14days after the completion of the cell fusion. When the culture solutionturned yellow (about 1×10⁵ cells/ml), the supernatants were recoveredand the antibody titer was assayed.

EXAMPLE 7 Screening of Hybridoma Cell

To the anti-mouse immunoglobulin antibody-bound microplate were added 50μl of buffer E and 50 μl of the hybridoma culture supernatant, followedby reaction at room temperature for 6 hours. After the plate was washedwith PBS, 100 μl of the HRP-labeled PACAP38NH₂ prepared in Example 4described above (diluted 200 times with buffer H) was added thereto,followed by reaction at 4° C. for 16 hours. After the plate was washedwith PBS, the enzyme activity on the solid phase was assayed by themethod described in Example 5-1 described above.

The supernatants of all of the 120 wells in which the proliferation ofthe cells was observed were thus examined. As a result, antibody titerwas detected in 18 wells.

EXAMPLE 8 Cloning

Of the wells which showed positive antibody activity, the hybridomacells contained in the wells of Nos. 44, 49, 97 and 113 were cloned bythe limiting dilution method. Namely, the hybridoma cells were floatedin RPMI 1640-20FCS so as to be contained in an amount of 1.5 cells/ml,and 2.0 ml thereof was poured into each well of a 96-well microplate(Nunk). In pouring, the thymocytes of BALB/C mice were added thereto asfeeder cells so as to be contained in an amount of 5×10⁵ cells/well.After about one week, the proliferation of the cells was observed. Theantibody titer of the supernatants was examined by the EIA described inExample 5. As a result, antibodies were produced in 28 clones of 30clones for the hybridoma cells of No. 44, in 47 clones of 50 clones forthe hybridoma cells of No. 49, in 49 clones of 50 clones for thehybridoma cells of No. 97 and in 48 clones of 50 clones for thehybridoma cells of No. 113. Of these clones, giving attention to clonePA-6N obtained from No. 44-2 and monoclonal antibody PA-6Na producedthereby, clone PA-1N obtained from No. 49-3 and monoclonal antibodyPA-1Na produced thereby, clone PA-2C obtained from No. 97-2 andmonoclonal antibody PA-2Ca produced thereby, and clone PA-5N obtainedfrom No. 113-5 and monoclonal antibody PA-5Na produced thereby, thefollowing experiments were made.

Similarly, the cell fusion experiment was conducted using the spleencells of another mouse during immunization. Giving attention also toclone PA-1C obtained from No. 28-12 and monoclonal antibody PA-1Caproduced thereby, and clone PA-3N obtained from No. 10-3 and monoclonalantibody PA-3Na produced thereby, the following experiment was carriedout.

EXAMPLE 9 Preparation of Large Amount of Monoclonal Antibodies

The mice which had been given 0.5 ml of mineral oil intraperitoneally orthe untreated mice (BALB/C) were injected intraperitoneally with 1 to3×10⁵ cells/mouse of the above-mentioned hybridoma cells, and then theantibody-containing ascites was recovered after 6 to 20 days.

EXAMPLE 10 Purification of Monoclonal Antibodies

The monoclonal antibodies were purified by a protein-A column or adiethylaminoethyl (DEAE)-cellulose column from the ascites obtained inExample 9 described above.

Namely, 6 ml of the ascites containing PA-1N was diluted with the sameamount of a binding buffer (pH 9.0, 1.5M glycine containing 3.5M NaCland 0.05% NAN₃). The resulting solution was subjected to a protein-ASepharose (Pharmacia) column which had been pre-equilibrated with thebinding buffer, and a specific antibody was eluted with an elutingbuffer (pH 3.0, 0.1M citrate buffer containing 0.05% NAN₃). By the aboveprocedures, 28 mg of the specific antibody was obtained.

Similarly, 23 mg of a specific antibody was obtained from 5 ml of theascites containing PA-5N, 13 mg of a specific antibody was obtained from7.5 ml of the ascites containing PA-6N, and 45 mg of a specific antibodywas obtained from 14 ml of the ascites containing PA-1C.

On the other hand, a saturated ammonium sulfate solution was added to 20ml of the ascites containing PA-3N to a final concentration of 45% forsalt precipitation, followed by centrifugation (20,000 g, 30 minutes).The precipitate fraction was dialyzed against 0.02M borate buffer (pH 7)containing 0.15M NaCl (hereinafter referred to as BBS), and furtherdialyzed against 0.01M phosphate buffer containing 0.01M NaCl. Theantibody fraction was loaded on a DEAE cellulose column (DE-52, Wattman,2.5 cm in diameter×10 cm), and eluted by a linear concentration gradient(0.01M-0.35M) of 100 ml of NaCl. By the above procedures, 136 mg of aspecific antibody was obtained.

Similarly, 57 mg of a specific antibody was obtained from 7.5 ml of theascites containing PA-2C.

EXAMPLE 11 Determination of Class and Subclass of Monoclonal Antibody

Into each well of a 96-well microplate was poured 100 μl of 0.1Mcarbonate buffer (pH 9.6) containing 5 μg/ml of PACAP38NH₂ preparedabove, and the microplate was allowed to stand at 4° C. for 24 hours.The excess binding sites of the wells were blocked with Blockaceaccording to the method described in Example 5-1 to prepare a PACAP38NH₂ -bound plate. Then, each of supernatants of PA-1N, PA-3N, PA-5N,PA-6N, PA-2C and PA-1C was added to each well of the plate in an amountof 100 μl, followed by reaction at room temperature for 3 hours. Then,the class and subclass were examined by the enzyme-linked immunosorbentassay (ELISA) using an isotype typing kit (Mouse-Typer™ Sub-IsotypingKit, Bio RAD). As a result, PA-1Na, PA-6Na, PA-2Ca and PA-1Ca belongedto IgGl, κ, PA-5Na belonged to IgG2a, κ, and PA-3Na belonged to IgG2b,κ.

EXAMPLE 12 Preparation of F(ab')₂ Fraction

PA-6Na described in Example 10 was concentrated to 8 mg/500 μl by aCollodion bag (Emuesu Kiki), and then dialyzed against 0.1M acetatebuffer containing 0.1M NaCl. To the resulting antibody solution wasadded 0.4 mg of pepsin (crystallized twice, Sigma), followed by reactionat 37° C. for 16 hours. Then, the F(ab')₂ fraction was purified by anFPLC (Pharmacia) using a Superrose 12 column equilibrated with 0.1Mphosphate buffer (pH 6.8).

By a similar technique, 0.445 mg of pepsin was added to 8.9 mg of PA-1Cadescribed in Example 10 to prepare the F(ab') fraction.

EXAMPLE 13 Preparation of HRP-Labeled Anti-PACAP Monoclonal Antibodies

(1) PA-6Na F(ab')₂ -HRP

To 1 ml of a solution containing 2.2 mg (22 nmoles)/ml of the PA-6NaF(ab')₂ fraction described in Example 12 was added 50 μl of a DMFsolution containing 260 nmoles of GMBS, followed by reaction at roomtempedrature for 40 minutes. The reaction solution was fractionated on aSephadex G-25 column [1 cm in diameter×30 cm, eluent: 0.1M phosphatebuffer (pH 6.7)] to obtain a maleimide group-introduced F(ab')₂fraction. With 1.5 mg of the resulting F(ab')₂ fraction was mixed 5.5 mgof SH group-introduced HRP prepared by the method described in Example4-1, and the reaction product was concentrated to about 0.3 ml by acollodion bag, followed by standing at 4° C. for 16 hours. The reactionsolution was loaded on an Ultrogel AcA34 column (10 mm in diameter×40mm) to purify an F(ab')₂ -HRP complex fraction. It was confirmed fromthe absorbance at 280 nm and 403 nm that HRP was introduced in an amountof 2.4 molecules/molecule of F(ab')₂.

(2) PA-1Ca F(ab')₂ -HRP

In a similar manner, an F(ab')₂ -HRP complex was prepared by using 2.9mg of the PA-1Ca F(ab')₂ fraction described in Example 12.

(3) PA-2Ca IgG-HRP

To 6.4 mg (43 nmoles) of the PA-2Ca purified fraction described inExample 10 was added 15-fold moles of GMBS, followed by introduction ofa maleimide group. Then, the resulting product was reacted with SHgroup-introduced HRP in a similar manner to prepare a marker into whichHRP was introduced in an amount of 2.4 molecules/molecule of IgG.

EXAMPLE 14 Competitive Method-EIA

(1) Competitive Method-EIA Using PA-1Na

To the anti-mouse immunoglobulin antibody-bound microplate described inExample 5 were added 50 μl of a PA-1N culture supernatant diluted 50times with buffer H and 50 μl of a buffer H solution of PACAP or apartial peptide of PACAP, such as PACAP38NH₂, PACAP27NH₂, PACAP(4-27),PACAP(1- 13), PACAP(14-38)NH₂, PACAP(31-38)NH₂ or VIP, followed byreaction at room temperature for 2 hours. Then, 50 μl of HRP-labeledPACAP38NH2 described in Example 4-1 (diluted 100 times with buffer H)was added thereto, followed by reaction at 4° C. for 16 hours. Afterreaction, the plate was washed with PBS, and then the enzyme activity onthe solid phase was assayed by the method described in Example 5. Theresults are shown in FIG. 3(a). In the drawing, -o-, - -, - -, - -, -Δ-,-□- and -X- indicate PACAP38NH₂, PACAP27NH₂, PACAP(4-27)OH,PACAP(1-13)OH, PACAP(14-38)NH₂, PACAP(31-38)NH₂ and a standard curve ofVIP, respectively.

As shown in FIG. 3(a), PA-1Na reacts with PACAP38NH₂, PACAP27NH₂,PACAP(1-13)OH and PACAP(4-27)OH, but does not react with PACAP(14-38)NH₂and PACAP(31-38)NH₂. PA-1Na does not react with VIP either (the crossreactivity to PACAP38NH₂ is 0.1% or less). These results reveal thatPA-1Na is an antibody belonging to class Ia which recognizes theN-terminal portion of PACAP38NH₂.

(2) Competitive Method-EIA Using PA-5Na

A competitive method-EIA using PA-5Na was carried out by the methoddescribed described in Example 14-(1). A culture supernatant of PA-5Nwas diluted 70 times. The results are shown in FIG. 3(c). PA-5Na reactswith PACAP38NH₂, PACAP27NH₂, PACAP(1-13)OH and PACAP(4-27)OH, but doesnot react with PACAP(14-38)NH₂ and PACAP(31-38)NH₂. PA-5Na does notreact with VIP either (the cross reactivity is 0.1% or less). Theseresults reveal that PA-5Na is an antibody belonging to class Ia whichrecognizes the N-terminal portion of PACAP38NH₂.

PA-1Na is different from PA-5Na in cross reactivity (to PACAP38NH₂) withPACAP(1-13)OH, and the cross reactivity of the former is at least 10times stronger than that of the latter.

(3) Competitive Method-EIA Using PA-3Na

A competitive method-EIA using PA-3Na was carried out by the methoddescribed in Example 14-(1). A culture supernatant of PA-3N was diluted50 times. The results are shown in FIG. 3(b). PA-3Na reacts withPACAP38NH₂, PACAP27NH₂, PACAP(1-13)OH and PACAP(4-27)OH, but does notreact with PACAP(14-38)NH₂ and pacap(31-38)NH₂. On the other hand,PA-3Na shows a cross reactivity of 1% with VIP (to PACAP38NH₂). Theseresults reveal that PA-5Na is an antibody belonging to class Ib whichrecognizes the N-terminal portion of PACAP38NH₂.

(4) Competitive Method-EIA Using PA-6Na

A competitive method-EIA using PA-6Na was carried out by the methoddescribed in Example 14-(1). A culture supernatant of PA-6N was diluted40 times. The results are shown in FIG. 3(d). Pa-6Na reacts withPACAP38NH₂, PACAP27NH₂ and PACAP(4-27)OH, but does not react withPACAP(1-13)OH, PACAP(14-38)NH₂ and PACAP(31-38)NH₂. PA-6Na does notreact with VIP either (the cross reactivity to PACAP38NH₂ is 0.1% orless). These results reveal that PA-6Na is an antibody belonging toclass II which recognizes the region from the N-terminal portion to thecentral portion of PACAP38NH₂.

(5) Competitive Method-EIA Using PA-2Ca

A competitive method-EIA using PA-2Ca was carried out by the methoddescribed described in Example 14-(1). A culture supernatant of PA-2Cwas diluted 340 times. The results are shown in FIG. 3(e). PA-1Ca reactswith PACAP38NH₂ and PACAP(14-38)NH₂, but does not react with PACAP27NH₂,PACAP(4-27)OH PACAP(1-13)OH and PACAP(31-38)NH₂. PA-2Ca does not reactwith VIP either (the cross reactivity to PACAP38NH₂ is 0.1% or less).These results reveal that PA-2Ca is an antibody belonging to class IIIwhich recognizes the region from the C-terminal portion to the centralportion of PACAP38NH₂.

(6) Competitive Method-EIA Using PA-1Ca

A competitive method-EIA using PA-1Ca was carried out by the methoddescribed described in Example 14-(1). A culture supernatant of PA-1Cwas diluted 35 times. The results are shown in FIG. 3(f). PA-1Ca reactswith PACAP38NH₂, PACAP(14-38)NH₂ AND pacap(31-38)NH₂, but does not reactwith PACAP27NH₂, PACAP(4-27)OH and PACAP(1-13)OH. PA-6Na does not reactwith VIP either (the cross reactivity to PACAP38NH₂ is 0.1% or less).These results reveal that PA-2Ca is an antibody belonging to class IVwhich recognizes the C-terminal portion of PACAP38NH₂.

At least 400, 100, 200, 200, 20 or 200 pg/well (a PACAP concentrationgiving B/BO=80%) of PACAP38NH₂ could be detected by the competitivemethod EIA using PA-1Na, PA-5Na, PA-3Na, PA-6Na, PA-2Ca or PA-1Ca.

EXAMPLE 15 Sandwich Method-EIA

(1) Sandwich Method-EIA Using PA-6Na F(ab')₂ -HRP

Into each well of a 96-well microplate was poured 100 μl of 0.1Mcarbonate buffer (pH 9.6) containing 15 μg/ml of purified monoclonalantibody PA-1Na, PA-3Na, PA-5Na, PA-6Na, PA-2Ca or PA-1Ca described inExample 10, and the plate was allowed to stand at 4° C. for 24 hours.The excess binding sites of the wells were inactivated by adding 300 μlof Blockace diluted 4 times with PBS.

To each well of the plate thus prepared was added 100 μl of a standardsolution of PACAP38NH₂ diluted with buffer E, followed by reaction at 4°C. for 24 hours. After washing with PBS, 100 μl of HRP-labeled PA-γNaF(ab')₂ prepared in Example 13-(1) described above (diluted 100 timeswith buffer C) was added thereto, followed by reaction at 4° C. for 24hours. After washing with PBS, the enzyme activity on the solid phasewas assayed by the method described in Example 5. The results are shownin FIG. 4.

In the sandwich method-EIA using PA-6Na (class II) F(ab')₂ -HRP, thesensitivity was highest when PA-2Ca (class III) was used as the antibodyfor solid phase, and at least 0.4 pg/well of PACAP38NH₂ could bedetected. When PA-1Ca (class IV) was used as the antibody for solidphase, 2 pg/well of PACAP38NH₂ could be detected, and when PA-5Na (classIa) was used, 40 pg/well of PACAP38NH₂ could be detected.

The above results reveal that when PA-6Na, an antibody of class II, isused as a marker, the sandwich-EIA can be established even if anantibody of any other class (including a class adjacent thereto inprimary arrangement) is used as the antibody for solid phase, andparticularly that the sandwich method-EIA using PA-2Ca, an antibody ofclass III, as the antibody for solid phase is highly sensitive.

(2) Sandwich Method-EIA Using PA-1Ca F(ab')₂ -HRP

A sandwich method-EIA using a microplate sensitized with each of variousantibodies described in the above item (1) and HRP-labeled PA-1CaF(ab')₂ described in Example 13-(2) was carried out by the methoddescribed in the above item (1). The results are shown in FIG. 5.

In the sandwich method-EIA using PA-1Ca (class IV) F(ab')₂ -HRP, thesensitivity was highest when PA-3Na (class Ib) was used as the antibodyfor solid phase, and at least 1 pg/well of PACAP38NH₂ could be detected.When PA-1Na (class Ia), PA-5Na (class Ia) or PA-6Na (class II) was usedas the antibody for solid phase, at least 2 pg/well of PACAP38NH₂ couldbe detected. Further, even when and antibody of class III adjacent inprimary arrangement was used, at least 8 pg/well of PACAP38NH₂ wasdetected.

The above results reveal that when PA-1Ca, an antibody of class IV, isused as a marker, the sandwich-EIA can be established even if anantibody of any other class (including a class adjacent thereto inprimary arrangement) is used as the antibody for solid phase, andparticularly that the sandwich method-EIA using PA-3Na, an antibody ofclass Ib, as the antibody for solid phase is highly sensitive.

(3) Sandwich Method-EIA Using PA-2Ca IgG-HRP

A sandwich method-EIA using a microplate sensitized with each of variousantibodies described in the above item (1) and HRP-labeled PA-2Ca IgGdescribed in Example 13-(3) was carried out by the method described inthe above item (1). The results are shown in FIG. 6.

In the sandwich method-EIA using PA-2Ca (class III) IgG-HRP, thesensitivity was highest when PA-3Na (class Ib) was used as the antibodyfor solid phase, and at least 2 pg/well of PACAP38NH₂ could be detected.When PA-1Na, PA-5Na or PA-6Na was used as the antibody for solid phase,4 pg/well of PACAP38NH₂ was detected, and when PA-1Ca was used, 80pg/well of PACAP38NH₂ was detected.

The above results reveal that when PA-2Ca, an antibody of class III, isused as a marker, the sandwich-EIA can be established even if anantibody of any other class (including a class adjacent thereto inprimary arrangement) is used as the antibody for solid phase, andparticularly that the sandwich method-EIA using PA-3Na, an antibody ofclass Ib, as the antibody for solid phase in highly sensitive.

EXAMPLE 16 Specificity of Sandwich Method-EIA

In a sandwich method-EIA using PA-2Ca described in Example 15-(1) as theantibody for solid phase and PA-6Na F(ab')₂ -HRP as the antibody forlabeling, the reactivity to PACAP38NH₂, VIP GRF and secretin wasexamined. The results are shown in FIG. 7.

In the drawing, -o-, -, - -, -Δ- and - - indicateconcentration-dependent curves of PACAP38NH₂, PACAP27NH₂, VIP, GRF andsecretin, respectively.

In the above assay, the cross reactivity to all of PACAP27NH₂, VIP, GRFand secretin is 0.001%. This reveals that the above assay is specificfor PACAP38NH₂.

EXAMPLE 17 Purification of Rabbit PACAP(11-27)NH₂ Antibody

An affinity solid phase for purifying rabbit PACAP(11-27)NH₂ wasprepared. Namely, 4.5 mg of [Cys¹⁰ ]PACAP(11-27)NH₂ was dissolved in 20ml of 0.1M sodium hydrogencarbonate containing 0.5M NaCl, and reactedwith 3 g of CNBr-activated Sepharose 4B at room temperature for 3 hours.Then, after unreacted active groups were treated with 0.1MTris-hydrochloric acid buffer (pH 8), the resulting product wasdispersed in PBS and charged into a column.

8 ml of rabbit PACAP(11-27)NH₂ antiserum 1C, 8 ml of 2C and 16 ml of 3Cin which high antibody activity was observed (see FIG. 2) were mixedwith one another, and 32 ml of PBS was added thereto. Then, 52 ml ofsaturated ammonium sulfate was slowly added thereto with stirring,followed by centrifugation at 12,000×g for 20 minutes. The precipitatewas dissolved in 25 ml of borate buffer (pH 8) containing 0.15M NaCl(BBS), followed by dialysis against BBS at 4° C. for 2 days. Afterdialysis, the resulting solution was loaded on the above-mentionedcolumn and thoroughly washed with BBS. Then, specific antibodies wereeluted with 0.1M acetate buffer (pH 4.5) containing 0.5M NaCl andfurther with 0.05M glycine-hydrochloric acid buffer (pH 2.0) containing0.1M NaCl. As a result, 5.4 mg and 6.7 mg of the specific antibodieswere obtained in fractions eluted at pH 4 and pH 2, respectively.

EXAMPLE 18 Preparation of Anti-PACAP(11-27)NH₂ Fab'-HRP

An Fab'-peroxidase marker was prepared from the anti-PACAP(11-27)NH₂antibody described in Example 17 according to the method of Ishikawa etal. [J. Appl. Biochem. 6, 56-63 (1984)].

Namely, 5.6 mg of the specific antibody was dissolved in 0.1M acetatebuffer (pH 4.5), and 160 μg of pepsin (crystallized twice, Sigma) wasadded thereto, followed by reaction at 37° C. for 20 hours. Then, thereaction product was subjected to an FPLC (Pharmacia) using a Superose12 column equilibrated with 0.1M acetate buffer (pH 5) to obtain 2.2 mgof an F(ab')₂ fraction. β-Mercaptoethylamine was added to this fractionto a final concentration of 20 mM, and the resulting solution wasallowed to stand at 37° C. for 90 minutes. Then, the reaction solutionwas separated on a Sephadex G-25 Column equilibrated with 0.1M phosphatebuffer (pH 6.0) containing 5 mM EDTA to obtain an Fab' fraction.

On the other hand, 6 mg of maleimidated HRP prepared according to themethod described in Example 4-2 and the total amount of theabove-mentioned anti-PACAP(11-27)NH Fab' fraction were mixed and reactedwith each other at 4° C. for 1 days. Then, the reaction product wasfractionated on an Ultrogel AcA44 column equilibrated with 0.1Mphosphate buffer to purify HRP-labeled anti-PACAP(11-27)NH₂ Fab'.

EXAMPLE 19 Sandwich Method-EIA for Assaying PACAP27NH₂

(1) To a microplate on which PA-1Na described in Example 15 was fixed,100 μl of a standard solution of PACAP27NH₂, PACAP(11-27)OH orPACAP38NH₂ was added, followed by reaction at 4° C. for 24 hours. Afterwashing with PBS, HRP-labeled anti-PACAP(11-27)NH₂ Fab' described inExample 18 (diluted 400 times with buffer C) was added thereto andreacted at 4° C. for 24 hours. After washing with PBS, the enzymeactivity on the solid phase was assayed by the method described inExample 5-2. The results are shown in FIG. 8.

In the drawing, - -, - -, - - and -o- indicate PACAP27NH₂,PACAP(11-27)OH, PACAP38NH₂ and VIP, respectively. The results shown inFIG. 8 reveal that 0.2 pg/well of PACAP27NH₂ can be detected by thisassay with a cross reactivity of 11.5% with PACAP(11-27)OH and with across reactivity of 0.97% by weight ratio or 1.3% by mole ratio withPACAP38NH₂.

(2) Using a microplate on which PA-3Na described in Example 15 was fixedand HRP-labeled PACAP(11-27)NH₂ Fab' described in Example 18, a sandwichmethod-EIA was carried out by the method described above. The resultsare shown in FIG. 9. These results reveal that 0.2 pg/well of PACAP27NH₂can be detected by this assay with a cross reactivity of 11% withPACAP(11-27)OH and with a cross reactivity of 0.22% by weight ratio or0.31% by mole ratio with PACAP38NH₂.

(3) Using a microplate on which PA-5Na described in Example 15 was fixedand HRP-labeled PACAP(11-27)NH₂ Fab' described in Example 18, a sandwichmethod-EIA was carried out by the method described above. The resultsare shown in FIG. 10. These results reveal that 0.2 pg/well ofPACAP27NH₂ can be detected by this assay with a cross reactivity of 4.1%with PACAP(11-27)OH and with a cross reactivity of 0.40% by weight ratioor 0.56% by mole ratio with PACAP38NH₂.

(4) Using a microplate on which PA-6Na described in Example 15 was fixedand HRP-labeled PACAP(11-27)NH₂ Fab' described in Example 18, a sandwichmethod-EIA was carried out by the method described above. The resultsare shown in FIG. 11. These results reveal that 0.8 pg/well ofPACAP27NH₂ can be detected by this assay with a cross reactivity of 5.4%with PACAP(11-27)OH and with a cross reactivity of 3.6% by weight ratioor 5.00% by mole ratio with PACAP38NH₂.

All of the assays only exhibited a cross reactivity of 0.001% or lesswith VIP.

From the above results, PACAP27NH₂ can be detected by the use of theseassays with a cross reactivity of 0.22 to 3.6% by weight ratio or with across reactivity of 0.31 to 5.0% by mole ratio with PACAP38NH₂. It istherefore possible to fractionate and determine PACAP27NH₂ andPACAP38NH₂ by combinations of these assays and the assay described inExample 16.

EXAMPLE 20 Examination of Neutralization Activity of Anti-PACAPAntibodies

Rat adrenal brown cytoma strain PC-12h (supplied by Dr. Hatanaka,Protein Laboratory, Osaka University) was disseminated at a rate of5×10⁴ cells/well on a 48-well multi-well plate (Sumitomo Bakelite)treated with collagen, and incubated in Dulbecco's modified Eagle'smedium (DMEM) containing 10% FCS for 7 to 10 days. The medium of theplate was exchanged with Hnak's balanced salt solution (HBSS) containing0.05% BSA, followed by incubation for 30 minutes. Then, PACAP38NH₂(final concentration: 2 nM) was added thereto which had previously beenreacted with each of the anti-PACAP antibodies (final concentration: 2,20 or 200 nM) at 4° C. for 1 hour. After additional incubation for 2hours, the concentration of cAMP contained in the culture supernatantwas measured with a cAMP measuring kit (Amersham). The results are shownin FIG. 12. In the drawing, -o-, -Δ-, -□-, - -, - and - - indicatePA-1Na, PA-3Na, PA-5Na, PA-6Na, PA-2Ca and PA-1Ca, respectively. Theseresults reveal that four kinds of these six kinds of monoclonalanti-PACAP antibodies have neutralization activity to PACAP38NH₂ and theorder of its strength is PA-2Ca (an antibody of class III)>PA-1Na (anantibody of class Ia)>PA-5Na (an antibody of class Ia)>PA-3Na (anantibody of class Ib).

The use of the various monoclonal antibodies of the present inventionwhich recognized the continuous sites in primary arrangement of PACAPrevealed immunochemical properties of PACAP. By using these antibodies,it is possible to establish assay systems using various competitive orsandwich methods different in specificity for PACAP or its relatedpeptides. In particular, it becomes possible to fractionate anddetermine PACAP38NH₂ and PACAP27NH₂ with high sensitivity bycombinations of various sandwich methods.

EXAMPLE 21 Examination of Reactivity of Anti-PACAP Monoclonal Antibodieswith Human PACAP Precursor

Escherichia coli cells having plasmid pTS401 in which human PACAPprecursor genes were integrated were cultivated in 10 ml of M9 medium(containing 0.1% NZ amine, 0.4% glucose, 50 μg/ml ampicillin and 25μg/ml chloramphenicol) at 37° C. When the cells were proliferated tohave an absorbance of 0.7 at 600 nm, isopropyl-β-D-thiogalactopyranosidewas added thereto, and cultivation was further continued for 3 hours,followed by centrifugation at 5,000 g for 10 minutes to collect thecells. As a control, the cells were collected just before addition ofisopropyl-β-D-thiogalactopyranoside.

To the cells, 1 ml of SDS-containing sample buffer of Laemmli was added,and the mixture was boiled at 100° C. for 5 minutes, followed byelectrophoresis on 16% polyacrylamide gel. The protein was electricallytransferred to nitrocellulose filters by the western blotting method,and each of anti-PACAP mouse monoclonal antibodies PA-1Na, PA-1Ca andPA-2Ca was reacted with the protein on each filter. Then, the secondaryantibody (anti-mouse IgG-peroxidase, Cappel.) was reacted therewith. Theband of the desired protein was stained and fixed by a POD immunostainset (Wako Pure Chemical Industries). The bands reacting with anti-PACAPmouse monoclonal antibodies PA-1Na and PA-2Ca were observed in thevicienity of a molecularf weight of about 18,000 daltons. The bandsapproximately agree with molecular weight of human PACAP precursordeduced from cDNA coding for human PACAP. This fact reveals thatanti-PACAP mouse monoclonal antibodies PA-1Na and PA-2Ca react withhuman PACAP precursor.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       HisSerAs pGlyIlePheThrAspSerTyrSerArgTyrArgLysGln                             151015                                                                        MetAlaValLysLysTyrLeuAlaAlaValLeuGlyLysArgTyrLys                               202530                                                                       GlnArgValLysAsnLys                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       HisSerAspGlyIlePheThrAspSerTyrSerArgTyrArgLysGln                              151015                                                                        MetAlaValLysLysTyrLeuAlaAl aValLeu                                            2025                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       HisSerAspGlyIlePhe ThrAspSerTyrSerArgTyr                                      1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       Gl yIlePheThrAspSerTyrSerArgTyrArgLysGlnMetAlaVal                             151015                                                                        LysLysTyrLeuAlaAlaValLeu                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ArgLysGlnMetAlaValLysLysTyrLeuAlaAlaValLeuGlyLys                              1 51015                                                                       ArgTyrLysGlnArgValLysAsnLys                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrLysGlnArgValLysAsnLys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerArgTyrArgLysGlnMetAlaValLysLysTyrLeuAlaAlaVal                              151015                                                                        Leu                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 176 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetThrMetCysSerGlyAlaArgLeuAlaLeuLeuValTyrGlyIle                              15 1015                                                                       IleMetHisSerSerValTyrSerSerProAlaAlaAlaGlyLeuArg                              202530                                                                        PheProGlyIleArgProGlu GluGluAlaTyrGlyGluAspGlyAsn                             354045                                                                        ProLeuProAspPheGlyGlySerGluProProGlyAlaGlySerPro                              5055 60                                                                       AlaSerAlaProArgAlaAlaAlaAlaTrpTyrArgProAlaGlyArg                              65707580                                                                      ArgAspValAlaHisGlyIleLe uAsnGluAlaTyrArgLysValLeu                             859095                                                                        AspGlnLeuSerAlaGlyLysHisLeuGlnSerLeuValAlaArgGly                              100 105110                                                                    ValGlyGlySerLeuGlyGlyGlyAlaGlyAspAspAlaGluProLeu                              115120125                                                                     SerLysArgHisSerAspGl yIlePheThrAspSerTyrSerArgTyr                             130135140                                                                     ArgLysGlnMetAlaValLysLysTyrLeuAlaAlaValLeuGlyLys                              145150 155160                                                                 ArgTyrLysGlnArgValLysAsnLysGlyArgArgIleAlaTyrLeu                              165170175                                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TyrAlaAspAlaIlePheThrAsnSerTyrArgLysValLeuGlyGln                              15 1015                                                                       LeuSerAlaArgLysLeuLeuGlnAspIleMetSerArgGlnGlnGly                              202530                                                                        GluSerAsnGlnGluArgGlyA laArgAlaArgLeu                                         3540                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      HisSerAspGlyThr PheThrSerGluLeuSerArgLeuArgGluGly                             151015                                                                        AlaArgLeuGlnArgLeuLeuGlnGlyLeuVal                                             20 25                                                                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      HisSerAspAlaValPheThrAspAsnTyrThrArgLeuArgLysGln                               151015                                                                       MetAlaValLysLysTyrLeuAsnSerIleLeuAsn                                          2025                                                                      

What is claimed is:
 1. The hybridoma cell line FERM BP-2811.
 2. Thehybridoma cell line FERM BP-2813.
 3. The hybridoma cell line FERMBP-2814.
 4. The hybridoma cell line FERM BP-2815.
 5. The hybridoma cellline FERM BP-2816.
 6. Monoclonal antibody PA-1Na, produced by thehybridoma cell line of claim
 1. 7. Monoclonal antibody PA-5Na, producedby the hybridoma cell line of claim
 2. 8. Monoclonal antibody PA-6Na,produced by the hybridoma cell line of claim
 3. 9. Monoclonal antibodyPA-2Ca, produced by the hybridoma cell line of claim
 4. 10. Monoclonalantibody PA-1Ca, produced by the hybridoma cell line of claim
 5. 11. Apolyclonal antibody which specifically binds to a C-terminal peptide ofPACAP27 represented by SEQ ID NO:7.